Measuring the elasticity of liquid–liquid phase separation droplets with biomembrane force probe

Min Sun; Hui Chen; Qinghua Ji; Jianhui Xiao; Yanzhe Hou; Jizhong Lou

doi:10.52601/bpr.2022.210038

Volume 8 Issue 2

Apr. 2022

Turn off MathJax

Article Contents

Article Navigation > Biophysics Reports > 2022 > 8(2): 68-79

Min Sun, Hui Chen, Qinghua Ji, Jianhui Xiao, Yanzhe Hou, Jizhong Lou. Measuring the elasticity of liquid–liquid phase separation droplets with biomembrane force probe[J]. Biophysics Reports, 2022, 8(2): 68-79. doi: 10.52601/bpr.2022.210038

Citation:

Min Sun, Hui Chen, Qinghua Ji, Jianhui Xiao, Yanzhe Hou, Jizhong Lou. Measuring the elasticity of liquid–liquid phase separation droplets with biomembrane force probe[J]. Biophysics Reports, 2022, 8(2): 68-79. doi: 10.52601/bpr.2022.210038

Citation:

PDF (18644 KB)

Measuring the elasticity of liquid–liquid phase separation droplets with biomembrane force probe

doi: 10.52601/bpr.2022.210038

Min Sun^{1, 2, #},
Hui Chen^{1, #},
Qinghua Ji^{1, 2},
Jianhui Xiao^{1, 2},
Yanzhe Hou³,
Jizhong Lou^{1, 2
,
,}

1.
Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
2.
University of Chinese Academy of Sciences, Beijing 100049, China
3.
School of Basic Medical Pharmacology, Lanzhou University, Lanzhou 730000, China

Funds: The authors would like to thank Dr. Wei Chen for BFP instrumentation and Dr. Hong Zhang, Dr. Pilong Li for helpful discussions on the material properties of LLPS droplets. This work was supported by grants from the National Natural Science Foundation of China (32090044 and 11672317), the National Basic Research Program of China (2019YFA0707001), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB37020102)

More Information

Corresponding author: jlou@ibp.ac.cn (J. Lou)
Received Date: 02 August 2021
Accepted Date: 08 February 2022

Available Online: 25 March 2022

Publish Date: 22 April 2022

Abstract

Abstract

Numerous biomacromolecules undergo liquid–liquid phase separation (LLPS) inside living cells and LLPS plays important roles in their functions. The droplets formed by LLPS molecules are complex fluids and their behavior follows fluid mechanics, thus studies on rheological and material properties are required to gain full insight into the biophysical mechanism of these droplets. Biophysical force spectroscopy techniques are particularly useful in this aspect. Indeed, atomic force microscopy and optical tweezers have been used to quantify the elasticity and the viscoelasticity of LLPS droplets. The Biomembrane Force Probe (BFP) is a single-molecule technique designed to investigate liquid-like objects and is more suitable to quantify the material properties of LLPS droplets, but its usage on LLPS droplets is not yet described. Here we present an experimental protocol to measure the Young’s modulus of LLPS droplets using BFP, we believe that the application of BFP on phase separation studies can be expanded and will be very helpful in deciphering the underlying principles of LLPS.
- Phase separation,
- Force spectroscopy,
- Young’s modulus

# Min Sun and Hui Chen contributed equally to this work.

FullText(HTML)

References(28)

References

[1]	An C, Hu W, Gao J, Ju BF, Obeidy P, Zhao YC, Tu X, Fang W, Ju LA, Chen W (2020) Ultra-stable biomembrane force probe for accurately determining slow dissociation kinetics of PD-1 blockade antibodies on single living cells. Nano Lett 20(7): 5133−5140 doi: 10.1021/acs.nanolett.0c01360
[2]	Banani SF, Lee HO, Hyman AA, Rosen MK (2017) Biomolecular condensates: organizers of cellular biochemistry. Nat Rev Mol Cell Biol 18(5): 285−298 doi: 10.1038/nrm.2017.7
[3]	Bracha D, Walls MT, Wei MT, Zhu L, Kurian M, Avalos JL, Toettcher JE, Brangwynne CP (2018) Mapping local and global liquid phase behavior in living cells using photo-oligomerizable seeds. Cell 175(6): 1467−1480.e1413 doi: 10.1016/j.cell.2018.10.048
[4]	Chen W, Evans EA, McEver RP, Zhu C (2008) Monitoring receptor-ligand interactions between surfaces by thermal fluctuations. Biophys J 94(2): 694−701 doi: 10.1529/biophysj.107.117895
[5]	Chen W, Lou J, Evans EA, Zhu C (2012) Observing force-regulated conformational changes and ligand dissociation from a single integrin on cells. J Cell Biol 199(3): 497−512 doi: 10.1083/jcb.201201091
[6]	Chen W, Lou J, Zhu C (2010) Forcing switch from short- to intermediate- and long-lived states of the alphaA domain generates LFA-1/ICAM-1 catch bonds. J Biol Chem 285(46): 35967−35978 doi: 10.1074/jbc.M110.155770
[7]	Chen Y, Ju LA, Zhou F, Liao J, Xue L, Su QP, Jin D, Yuan Y, Lu H, Jackson SP, Zhu C (2019) An integrin alphaIIbbeta3 intermediate affinity state mediates biomechanical platelet aggregation. Nat Mater 18(7): 760−769 doi: 10.1038/s41563-019-0323-6
[8]	Chen Y, Liu B, Ju L, Hong J, Ji Q, Chen W, Zhu C (2015) Fluorescence biomembrane force probe: concurrent quantitation of receptor-ligand kinetics and binding-induced intracellular signaling on a single cell. J Vis Exp (102): e52975. DOI: 10.3791/52975
[9]	Elbaum-Garfinkle S, Brangwynne CP (2015) Liquids, fibers, and gels: the many phases of neurodegeneration. Dev Cell 35(5): 531−532 doi: 10.1016/j.devcel.2015.11.014
[10]	Evangelopoulos AE, Glynos E, Madani-Grasset F, Koutsos V (2012) Elastic modulus of a polymer nanodroplet: theory and experiment. Langmuir 28(10): 4754−4767 doi: 10.1021/la2049037
[11]	Evans E, Ritchie K, Merkel R (1995) Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces. Biophys J 68(6): 2580−2587 doi: 10.1016/S0006-3495(95)80441-8
[12]	Jawerth L, Fischer-Friedrich E, Saha S, Wang J, Franzmann T, Zhang X, Sachweh J, Ruer M, Ijavi M, Saha S, Mahamid J, Hyman AA, Julicher F (2020) Protein condensates as aging Maxwell fluids. Science 370(6522): 1317−1323 doi: 10.1126/science.aaw4951
[13]	Jawerth LM, Ijavi M, Ruer M, Saha S, Jahnel M, Hyman AA, Julicher F, Fischer-Friedrich E (2018) Salt-dependent rheology and surface tension of protein condensates using optical traps. Phys Rev Lett 121(25): 258101. https://doi.org/10.1103/PhysRevLett.121.258101
[14]	Ju L (2019) Dynamic force spectroscopy analysis on the redox states of protein disulphide bonds. Methods Mol Biol 1967: 115−131
[15]	Ju L, Chen Y, Li K, Yuan Z, Liu B, Jackson SP, Zhu C (2017) Dual biomembrane force probe enables single-cell mechanical analysis of signal crosstalk between multiple molecular species. Sci Rep 7(1): 14185. https://doi.org/10.1038/s41598-017-13793-3
[16]	Ju L, Chen Y, Zhou F, Lu H, Cruz MA, Zhu C (2015a) Von Willebrand factor-A1 domain binds platelet glycoprotein Ibalpha in multiple states with distinctive force-dependent dissociation kinetics. Thromb Res 136(3): 606−612 doi: 10.1016/j.thromres.2015.06.019
[17]	Ju L, Lou J, Chen Y, Li Z, Zhu C (2015b) Force-Induced unfolding of leucine-rich repeats of glycoprotein Ibalpha strengthens ligand interaction. Biophys J 109(9): 1781−1784 doi: 10.1016/j.bpj.2015.08.050
[18]	Ju L, McFadyen JD, Al-Daher S, Alwis I, Chen Y, Tonnesen LL, Maiocchi S, Coulter B, Calkin AC, Felner EI, Cohen N, Yuan Y, Schoenwaelder SM, Cooper ME, Zhu C, Jackson SP (2018) Compression force sensing regulates integrin alphaIIbbeta3 adhesive function on diabetic platelets. Nat Commun 9(1): 1087. https://doi.org/10.1038/s41467-018-03430-6
[19]	Kuznetsova TG, Starodubtseva MN, Yegorenkov NI, Chizhik SA, Zhdanov RI (2007) Atomic force microscopy probing of cell elasticity. Micron 38(8): 824−833 doi: 10.1016/j.micron.2007.06.011
[20]	Liu B, Chen W, Evavold BD, Zhu C (2014) Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling. Cell 157(2): 357−368 doi: 10.1016/j.cell.2014.02.053
[21]	Murakami T, Qamar S, Lin JQ, Schierle GS, Rees E, Miyashita A, Costa AR, Dodd RB, Chan FT, Michel CH, Kronenberg-Versteeg D, Li Y, Yang SP, Wakutani Y, Meadows W, Ferry RR, Dong L, Tartaglia GG, Favrin G, Lin WL, Dickson DW, Zhen M, Ron D, Schmitt-Ulms G, Fraser PE, Shneider NA, Holt C, Vendruscolo M, Kaminski CF, St George-Hyslop P (2015) ALS/FTD mutation-induced phase transition of FUS liquid droplets and reversible hydrogels into irreversible hydrogels impairs RNP granule function. Neuron 88(4): 678−690 doi: 10.1016/j.neuron.2015.10.030
[22]	Poling-Skutvik R, Di X, Osuji CO (2020) Correlation of droplet elasticity and volume fraction effects on emulsion dynamics. Soft Matter 16(10): 2574−2580 doi: 10.1039/C9SM02394A
[23]	Shin Y, Chang YC, Lee DSW, Berry J, Sanders DW, Ronceray P, Wingreen NS, Haataja M, Brangwynne CP (2018) Liquid nuclear condensates mechanically sense and restructure the genome. Cell 175(6): 1481−1491.e1413 doi: 10.1016/j.cell.2018.10.057
[24]	Sibener LV, Fernandes RA, Kolawole EM, Carbone CB, Liu F, McAffee D, Birnbaum ME, Yang X, Su LF, Yu W, Dong S, Gee MH, Jude KM, Davis MM, Groves JT, Goddard WA, 3rd, Heath JR, Evavold BD, Vale RD, Garcia KC (2018) Isolation of a structural mechanism for uncoupling T cell receptor signaling from peptide-MHC binding. Cell 174(3): 672−687.e627 doi: 10.1016/j.cell.2018.06.017
[25]	Taylor NO, Wei MT, Stone HA, Brangwynne CP (2019) Quantifying dynamics in phase-separated condensates using fluorescence recovery after photobleaching. Biophys J 117(7): 1285−1300 doi: 10.1016/j.bpj.2019.08.030
[26]	Wu P, Zhang T, Liu B, Fei P, Cui L, Qin R, Zhu H, Yao D, Martinez RJ, Hu W, An C, Zhang Y, Liu J, Shi J, Fan J, Yin W, Sun J, Zhou C, Zeng X, Xu C, Wang J, Evavold BD, Zhu C, Chen W, Lou J (2019) Mechano-regulation of peptide-MHC class I conformations determines TCR antigen recognition. Mol Cell 73(5): 1015−1027.e1017 doi: 10.1016/j.molcel.2018.12.018
[27]	Zeng M, Chen X, Guan D, Xu J, Wu H, Tong P, Zhang M (2018) Reconstituted postsynaptic density as a molecular platform for understanding synapse formation and plasticity. Cell 174(5): 1172−1187.e1116 doi: 10.1016/j.cell.2018.06.047
[28]	Zhang H, Ji X, Li P, Liu C, Lou J, Wang Z, Wen W, Xiao Y, Zhang M, Zhu X (2020) Liquid-liquid phase separation in biology: mechanisms, physiological functions and human diseases. Sci China Life Sci 63(7): 953−985 doi: 10.1007/s11427-020-1702-x